In the first step in insulin action, insulin activates the insulin receptor tyrosine kinase. This leads to phosphorylation of tyrosine residues in multiple intracellular proteins. We have investigated this pathway in isolated rat adipocytes, a physiologically important target cell for insulin action. When recombinant epitope tagged GLUT4 glucose transporters are expressed in adipocytes, insulin stimulates the recruitment of these glucose transporters to the cell surface. When human insulin receptors are co-expressed in rat adipocytes, this leads to a marked increase in the number of epitope tagged glucose transporters on the cell surface. In contrast, Ile-1153 mutant insulin receptors are deficient in tyrosine kinase activity, and do not affect the number of epitope-tagged glucose transporters when co-expressed in rat adipocytes. Thus, tyrosine kinase activity is required for the ability of insulin receptors to mediate the effect of insulin to stimulate glucose transport in rat adipocytes. Thus, it is of interest to determine which intracellular proteins function downstream from the receptor tyrosine kinase in the pathway that leads to translocation of GLUT4 molecules to the cell surface. Insulin receptor substrate-1 (IRS-1) is among the best characterized substrates for phosphorylation by the insulin receptor. When IRS-1 levels were decreased by expression of an anti-IRS-1 ribozyme (designed to cleave rat IRS-1 mRNA), this impaired the ability of insulin to stimulate the translocation of epitope-tagged GLUT4 to the cell surface in isolated rat adipocytes. Furthermore, expression of human IRS-1 in rat adipocytes increased the number of epitope-tagged GLUT 4 molecules on the cell surface These observations suggest that IRS-1 mediates (at least in part) the effect of insulin to stimulate glucose transport. IRS-1 undergoes multi-site phosphorylation, thereby activating multiple SH2-domain-containing proteins that function in the insulin action pathway. Because expression of a dominant negative mutant of phosphatidyl inositol 3-kinase (PI-3-K) inhibits insulin action in rat adipocytes, this suggests that PI-3-K functions downstream from IRS-1.